Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
  • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
  • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/174—Vitamins
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
  • C11B3/00—Refining fats or fatty oils
  • C11B3/001—Refining fats or fatty oils by a combination of two or more of the means hereafter
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
  • C11B3/00—Refining fats or fatty oils
  • C11B3/12—Refining fats or fatty oils by distillation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/02—Oxygen as only ring hetero atoms
  • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein

Definitions

• the present invention is directed to nucleic acid and amino acid sequences and constructs, and methods related thereto.
• Isoprenoids are ubiquitous compounds found in all living organisms. Plants synthesize a diverse array of greater than 22,000 isoprenoids (Connolly and Hill (1992) Dictionary ofTerpenoids, Chapman and Hall, New York, NY). In plants, isoprenoids play essential roles in particular cell functions such as production of sterols, contributing to eukaryotic membrane architecture, acyclic polyprenoids found in the side chain of ubiquinone and plastoquinone, growth regulators like abscisic acid, gibberellins, brassinosteroids or the photosynthetic pigments chlorophylls and carotenoids.
• IPP isopentenyl diphosphate
• tocopherols A number of unique and interconnected biochemical pathways derived from the isoprenoid pathway leading to secondary metabolites, including tocopherols, exist in chloroplasts of higher plants. Tocopherols not only perform vital functions in plants, but are also important from mammalian nutritional perspectives. In plastids, tocopherols account for up to 40% of the total quinone pool.
• Tocopherols and tocotrienols are well known antioxidants, and play an important role in protecting cells from free radical damage, and in the prevention of many diseases, including cardiac disease, cancer, cataracts, retinopathy, Alzheimer's disease, andneurodegeneration, and have been shown to have beneficial effects on symptoms of arthritis, and in anti-aging.
• Vitamin E is used in chicken feed for improving the shelf life, appearance, flavor, and oxidative stability of meat, and to transfer tocols from feed to eggs. Vitamin E has been shown to be essential for normal reproduction, improves overall performance, and enhances immunocompetence in livestock animals. Vitamin E supplement in animal feed also imparts oxidative stability to milk products.
• the synthetic ⁇ -tocopherol has a vitamin E activity of 1J IU/mg.
• the worldwide market for raw refined tocopherols was $1020 million; synthetic materials comprised 85-88% of the market, the remaining 12-15% being natural materials.
• the best sources of natural tocopherols and tocotrienols are vegetable oils and grain products.
• most of the natural Vitamin E is produced from ⁇ -tocopherol derived from soy oil processing, which is subsequently converted to ⁇ -tocopherol by chemical modification ( ⁇ -tocopherol exhibits the greatest biological activity).
• the present invention is directed to D-1-deoxyxylulose 5 -phosphate reductoisomerase (dxr), and in particular to dxr polynucleotides and polypeptides.
• dxr D-1-deoxyxylulose 5 -phosphate reductoisomerase
• polynucleotides and polypeptides of the present invention include those derived from eukaryotic sources.
• one aspect of the present invention relates to isolated polynucleotide sequences encoding D-1-deoxyxylulose 5-phosphate reductoisomerase proteins.
• isolated nucleic acid sequences encoding dxr proteins from plant sources are provided.
• Another aspect of the present invention relates to oligonucleotides which include partial or complete dxr encoding sequences.
• constructs which can be used for transcription or transcription and translation (expression) of dxr.
• constructs are provided which are capable of transcription or transcription and translation in host cells.
• dxr in another aspect of the present invention, methods are provided for production of dxr in a host cell or progeny thereof.
• host cells are transformed or transfected with a DNA construct which can be used for transcription or transcription and translation of dxr.
• the recombinant cells which contain dxr are also part of the present invention.
• the present invention relates to methods of using polynucleotide and polypeptide sequences to modify the isoprenoid content of host cells, particularly in host plant cells. Plant cells having such a modified isoprenoid content are also contemplated herein.
• Figure 1 provides an amino acid alignment between the Arabidopsis dxr sequence and the E coli dxr sequence
• Figure 2 provides a schematic diagram of the isoprenoid pathway, both the mevalonate and non-mevalonate pathways.
• the present invention provides, ter alia, compositions and methods for altering (for example, increasing and decreasing) the isoprenoid levels and/or modulating their ratios in host cells.
• the present invention provides polynucleotides, polypeptides, and methods of use thereof for the modulation of isoprenoid content in host plant cells.
• Isoprenoids are derived from a 5- carbon building block, isopentenyl diphosphate (IPP), which is the universal isoprene unit and common isoprenoid precursor.
• Isoprenoids comprise a structurally diverse group of compounds that can be classified into two classes; primary and secondary metabolites (Chappell (1995) Annu Rev. Plant PhysioL Plant Mol. Biol. 46:521-547).
• Primary metabolites comprise those isoprenoids which are necessary for membrane integrity, photoprotection, orchestration of developmental programs, and anchoring biochemical functions to specific membrane systems.
• Such primary metabolites include, but are not limited to sterols, carotenoids, chlorophyll, growth regulators, and the polyprenol substituents of dolichols, quinones, and proteins.
• Secondary metabolites mediate important interactions between plants and the environment, but are not necessary to the viability of the plant. Secondary metabolites include, but are not limited to tocopherols, monoterpenes, sesquiterpenes, and diterpenes.
• the first reaction of the novel mevalonate-independent pathway is the condensation of (hvdroxyethyl)thiamin derived from pyruvate with the Cl aldehyde group of D-glyceraldehyde 3-phosphate to yield D-1-deoxyxylulose 5-phosphate (Broers (1994) Ph.D. Thesis Eidjustische Technische Hoch Engel, Zurich, Switzerland; Rohmer et al., (1996) J. Am. Chem. Soc. 118:2564-2566).
• D-1-deoxyxylulose In Escherichia coli, D-1-deoxyxylulose (most likely in the form of D-1-deoxyxylulose 5-phosphate) is efficiently incorporated into the prenyl-side chain of menaquinone and ubiquinone (Broers, (1994) supra; Rosa Putra et al., (1998) Tetrahedron Lett. 39:23-26). In plants, the inco ⁇ oration of D-1-deoxyxylulose into isoprenoids has also been reported (Zeidler et al., (1997) Z Naturforsch 52c: 15-23; Arigoni et al., (1997) Proc. Natl. Acad. Sci.
• D-1-deoxyxylulose has also been described as a precursor for the biosynthesis of thiamin and pyridoxol.
• D-l- deoxyxylulose is the precursor molecule of the contiguous five-carbon unit (C4'-C4- C5-C5'-C5") of thethiazole ring of thiamin in E. coli (Therisod et al., (l9Sl)Biochem. Biophys. Res. Comm. 98:374-379; David et al. (1981) J. Am. Chem. Soc.
• Figure 2 provides a schematic representation of the isoprenoid pathways.
• the enzyme 1-deoxy-D- xylulose 5-phosphate reductoisomerase batalyzing the conversion of 1-D-deoxy-D- xylulose 5-phosphate into 2-C-methyl-D-erythhyol 4-phosphate, has been recently cloned and characterized in E. coli (Takahashi et al., (1998) Proc. Natl. Acad. Sci. USA 95:99879-9884).
• the biosynthesis of 2-C-methyl-D-erythitol in plants by an intramolecular rearrangement of 1 -deoxy-D-xylulose 5-phosphate has recently been reported by Sagner et al. (1998) Tetrahedron Lett. 39:23-26 and Sagner et al. (1998) Chem Commun. 2:221-222.
• the present invention provides polynucleotide and polypeptide sequences involved in the production of 2-C-Methyl-D-erythritol-4- phosphate from 1- deoxyxylulose-5-phosphate, referred to as 1 -deoxy-D-xylulose 5-phosphate reductoisomerase or dxr. Also provided in the present invention are constructs and methods for the production of altered expression of dxr in host cells, as well as methods for the modification of the isoprenoid pathway, including modification of the biosynthetic flux through the isoprenoid pathway, and for the production of specific classes of isoprenoids in host cells.
• a first aspect of the present invention relates to isolated dxr polynucleotides.
• the polynucleotide sequences of the present invention include isolated polynucleotides that encode the polypeptides of the invention having a deduced amino acid sequence selected from the group of sequences set forth in the Sequence Listing and to other polynucleotide sequences closely related to such sequences and variants thereof.
• the invention provides a polynucleotide sequence identical over its entire length to each coding sequence as set forth in the Sequence Listing.
• the invention also provides the coding sequence for the mature polypeptide or a fragment thereof, as well as the coding sequence for the mature polypeptide or a fragment thereof in a reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, pro-, or prepro- protein sequence.
• the polynucleotide can also include non-coding sequences, including for example, but not limited to, non- coding 5' and 3' sequences, such as the transcribed, untranslated sequences.
• Polynucleotides of the present invention also include polynucleotides comprising a structural gene and the naturally associated sequences that control gene expression.
• the invention also includes polynucleotides of the formula:
• R 2 is a nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from the group set forth in the Sequence Listing and preferably those of SEQ ID NO: 1.
• R 2 is oriented so that its 5' end residue is at the left, bound to Ri, and its 3' end residue is at the right, bound to R 3 .
• Any stretch of nucleic acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
• the invention also relates to variants of the polynucleotides described herein that encode for variants of the polypeptides of the invention.
• Variants that are fragments of the polynucleotides of the invention can be used to synthesize full-length polynucleotides of the invention.
• Preferred embodiments are polynucleotides encoding polypeptide variants wherein 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues of a polypeptide sequence of the invention are substituted, added or deleted, in any combination. Particularly preferred are substitutions, additions, and deletions that are silent such that they do not alter the properties or activities of the polynucleotide or polypeptide.
• polynucleotide encoding a polypeptide of the invention and polynucleotides that are complementary to such polynucleotides. More preferable are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding a polypeptide of the invention and polynucleotides that are complementary thereto.
• polynucleotides at least 90% identical over their entire length are particularly preferred, those at least 95% identical are especially preferred.
• those with at least 97% identity are highly preferred and those with at least 98% and 99% identity are particularly highly preferred, with those at least 99% being the most highly preferred.
• Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptides encoded by the polynucleotides set forth in the Sequence Listing.
• the invention further relates to polynucleotides that hybridize to the above- described sequences.
• the invention relates to polynucleotides that hybridize under stringent conditions to the above-described polynucleotides.
• stringent conditions and “stringent hybridization conditions” mean that hybridization will generally occur if there is at least 95% and preferably at least 97% identity between the sequences.
• An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmon sperm DNA, followed by washing the hybridization support in OJx SSC at approximately 65°C.
• Other hybridization and wash conditions are well known and are exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, cold Spring Harbor, NY (1989), particularly Chapter 11.
• the invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set for in the Sequence Listing under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence or a fragment thereof; and isolating said polynucleotide sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers as described herein.
• polynucleotides of the invention can be used as a hybridization probe for RNA, cDNA, or genomic DNA to isolate full length cDNAs or genomic clones encoding a polypeptide and to isolate cDNA or genomic clones of other genes that have a high sequence similarity to a polynucleotide set forth in the Sequence Listing.
• Such probes will generally comprise at least 15 bases.
• Preferably such probes will have at least 30 bases and can have at least 50 bases.
• Particularly preferred probes will have between 30 bases and 50 bases, inclusive.
• each gene that comprises or is comprised by a polynucleotide sequence set forth in the Sequence Listing may be isolated by screening using a DNA sequence provided in the Sequence Listing to synthesize an oligonucleotide probe.
• a labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to identify members of the library which hybridize to the probe.
• synthetic oligonucleotides are prepared which correspond to the dxr sequences.
• the oligonucleotides are used as primers in polymerase chain reaction (PCR) techniques to obtain 5' and 3' terminal sequence of dxr genes.
• oligonucleotides of low degeneracy can be prepared from particular dxr peptides
• probes may be used directly to screen gene libraries for dxr gene sequences.
• screening of cDNA libraries inphage vectors is useful in such methods due to lower levels of background hybridization.
• a dxr sequence obtainable from the use of nucleic acid probes will show 60-70% sequence identity between the target dxr sequence and the encoding sequence used as a probe.
• lengthy sequences with as little as 50-60% sequence identity may also be obtained.
• the nucleic acid probes may be a lengthy fragment of the nucleic acid sequence, or may also be a shorter, oligonucleotide probe.
• longer nucleic acid fragments are employed as probes (greater than about 100 bp)
• one may screen at lower stringencies in order to obtain sequences from the target sample which have 20-50% deviation (i.e., 50-80% sequence homology) from the sequences used as probe.
• Oligonucleotide probes can be considerably shorter than the entire nucleic acid sequence encoding a dxr enzyme, but should be at least about 10, preferably at least about 15, and more preferably at least about 20 nucleotides. A higher degree of sequence identity is desired when shorter regions are used as opposed to longer regions. It may thus be desirable to identify regions of highly conserved amino acid sequence to design oligonucleotide probes for detecting and recovering other related dxr genes. Shorter probes are often particularly useful for polymerase chain reactions (PCR), especially when highly conserved sequences can be identified. (See, Gould, et al, PNAS USA (1989) 36:1934-1938.). Another aspect of the present invention relates to dxr polypeptides.
• polypeptides include isolated polypeptides set forth in the Sequence Listing, as well as polypeptides and fragments thereof, particularly those polypeptides which exhibit dxr activity and also those polypeptides which have at least 50%, 60% or 70% identity, preferably at least 80% identity, more preferably at least 90% identity, and most preferably at least 95% identity to a polypeptide sequence selected from the group of sequences set forth in the Sequence Listing, and also include portions of such polypeptides, wherein such portion of the polypeptide preferably includes at least 30 amino acids and more preferably includes at least 50 amino acids.
• Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
• identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences.
• Identity can be readily calculated by known methods including, but not limited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part i, Griffin, A.M.
• Computer programs which can be used to determine identity between two sequences include, but are not limited to, GCG (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology, 12: 76-80 (1994); Birren, et al., Genome Analysis, I: 543-559 (1997)).
• the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH, Bethesda. MD 20894; Altschul, S., et al., J. Mol. Biol, 215:403-410 (1990)).
• the well known Smith Waterman algorithm can also be used to determine identity.
• Parameters for polypeptide sequence comparison typically include the following:
• Parameters for polynucleotide sequence comparison include the following:
• the invention also includes polypeptides of the formula: XJR.) n -(R 2 )-(R 3 ) n -Y wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, Ri and R 3 are any amino acid residue, n is an integer between 1 and 1000, and R 2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from the group set forth in the Sequence Listing and preferably those encoded by the sequences provided in SEQ ID NO:2.
• R 2 is oriented so that its amino terminal residue is at the left, bound to Ri, and its carboxy terminal residue is at the right, bound to R 3 .
• Any stretch of amino acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
• Polypeptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising a sequence selected from the group of a sequence contained in the Sequence Listing set forth herein .
• polypeptides of the present invention can be mature protein or can be part of a fusion protein.
• a fragment is a variant polypeptide which has an amino acid sequence that is entirely the same as part but not all of the amino acid sequence of the previously described polypeptides.
• the fragments can be "free-standing" or comprised within a larger polypeptide of which the fragment forms a part or a region, most preferably as a single continuous region.
• Preferred fragments are biologically active fragments which are those fragments that mediate activities of the polypeptides of the invention, including those with similar activity or improved activity or with a decreased activity. Also included are those fragments that are antigenic or immunogenic in an animal, particularly a human.
• Variants of the polypeptide also include polypeptides that vary from the sequences set forth in the Sequence Listing by conservative amino acid substitutions, substitution of a residue by another with like characteristics. In general, such substitutions are among Ala, Val, Leu and He; between Ser and Thr; between Asp and Glu; between Asn and Gin; between Lys and Arg; or between Phe and Tyr. Particularly preferred are variants in which 5 to 10; 1 to 5; 1 to 3 or one amino acid(s) are substituted, deleted, or added, in any combination.
• Variants that are fragments of the polypeptides of the invention can be used to produce the co ⁇ esponding full length polypeptide by peptide synthesis. Therefore, these variants can be used as intermediates for producing the full-length polypeptides of the invention.
• polynucleotides and polypeptides of the invention can be used, for example, in the transformation of host cells, such as plant host cells, as further discussed herein.
• the invention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids within the mature polypeptide (for example, when the mature form of the protein has more than one polypeptide chain).
• Such sequences can, for example, play a role in the processing of a protein from a precursor to a mature form, allow protein transport, shorten or lengthen protein half-life, or facilitate manipulation of the protein in assays or production. It is contemplated that cellular enzymes can be used to remove any additional amino acids from the mature protein.
• a precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide.
• the inactive precursors generally are activated when the prosequences are removed. Some or all of the prosequences may be removed prior to activation.
• Such precursor protein are generally called proproteins.
• nucleotide sequences in recombinant DNA constructs to direct the transcription or transcription and translation (expression) of the dxr sequences of the present invention in a host cell.
• the expression constructs generally comprise a promoter functional in a host cell operably linked to a nucleic acid sequence encoding a dxr of the present invention and a transcriptional termination region functional in a host cell.
• Host cells of particular interest in the present invention include, but are not limited to, fungal cells, yeast cells, bacterial cells, mammalian cells, and plant cells.
• a first nucleic acid sequence is "operably linked” or “operably associated” with a second nucleic acid sequence when the sequences are so arranged that the first nucleic acid sequence affects the function of the second nucleic-acid sequence.
• the two sequences are part of a single contiguous nucleic acid molecule and more preferably are adjacent.
• a promoter is operably linked to a gene if the promoter regulates or mediates transcription of the gene in a cell.
• chloroplast and plastid specific promoters which are functional in plant cells, and have been described in the literature.
• chloroplast or plastid functional promoters and chloroplast or plastid operable promoters are also envisioned.
• One set of plant functional promoters are constitutive promoters such as the CaMV35S or FMV35S promoters that yield high levels of expression in most plant organs.
• Enhanced or duplicated versions of the CaMV35S and FMV35S promoters are useful in the practice of this invention (Odell, et al. (1985) Nature 313:810-812; Rogers, U.S. Patent Number 5,378, 619).
• nucleic acid sequences of the present invention from transcription initiation regions which are preferentially expressed in a plant seed tissue.
• seed preferential transcription initiation sequences include those sequences derived from sequences encoding plant storage protein genes or from genes involved in fatty acid biosynthesis in oilseeds.
• promoters include the 5' regulatory regions from such genes as napin (Kridl et al., Seed Sci. Res. 7:209:219 (1991)), phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, soybean ⁇ ' subunit of ⁇ -conglycinin (soy 7s, (Chen et al., Proc. Natl. Acad. Sci., 83:8560-8564 (1986))) and oleosin.
• CTP chloroplast transit peptides
• PTP plastid transit peptides
• the expression construct will additionally contain a gene encoding a transit peptide to direct the gene of interest to the plastid.
• the chloroplast transit peptides may be derived from the gene of interest, or may be derived from a heterologous sequence having a CTP. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 11544-11550; della-Cioppa et al.
• the constructs may contain the nucleic acid sequence which encodes the entire dxr protein, or a portion thereof.
• the entire dxr sequence is not required.
• Transcript termination regions may be provided in plant expression constructs of this invention as well.
• Transcript termination regions may be provided by the DNA sequence encoding the dxr or a convenient transcription termination region derived from a different gene source, for example, the transcript termination region which is naturally associated with the transcript initiation region. The skilled artisan will recognize that any convenient transcript termination region which is capable of terminating transcription in a plant cell may be employed in the constructs of the present invention.
• constructs may be prepared to direct the expression of the dxr sequences directly from the host plant cell plastid.
• constructs and methods are known in the art and are generally described, for example, in Svab, et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530 and Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917 and in U.S. Patent Number 5,693,507.
• constructs of the present invention can also be used in methods for altering the flux through the isoprenoid pathway with additional constructs for the expression of additional genes involved in the production of isoprenoids.
• sequences include, but are not limited to 1-deoxyxylulose 5-phosphate synthase.
• constructs of the present invention can be used in transformation methods with additional constructs providing for the expression of additional nucleic acid sequences encoding proteins in the production of specific isoprenoids, such as tocopherols, carotenoids, sterols, monoterpenes, sesquiterpenes, and diterpenes.
• nucleic acid sequences involved in the production of carotenoids and methods are described for example in PCT publication WO 99/07867.
• Nucleic acid sequences involved in the production of tocopherols include, but are not limited to gamma-tocpherol methyltransferase (Shintani, et al.
• a plant cell, tissue, organ, or plant into which the recombinant DNA constructs containing the expression constructs have been introduced is considered transformed, transfected, or transgenic.
• a transgenic or transformed cell or plant also includes progeny of the cell or plant and progeny produced from a breeding program employing such a transgenic plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a dxr nucleic acid sequence.
• Plant expression or transcription constructs having a dxr encoding sequence as the DNA sequence of interest for increased or decreased expression thereof may be employed with a wide variety of plant life.
• Particularly prefe ⁇ ed plants for use in the methods of the present invention include, but are not limited to: Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, ca ⁇ ot, cassava, cauliflower, celery, cherry, chicory, cilantro, citrus, Clementines, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew icama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, mango, melon
• temperate oilseed crops include, but are not limited to, rapeseed (Canola and High Erucic Acid varieties), sunflower, safflower, cotton, soybean, peanut, coconut and oil palms, and corn.
• rapeseed Canola and High Erucic Acid varieties
• sunflower safflower
• cotton cotton
• soybean peanut
• coconut peanut
• oil palms corn
• other DNA sequences may be required.
• this invention is applicable to dicotyledyons and monocotyledons species alike and will be readily applicable to new and/or improved transformation and regulation techniques.
• dxr constructs in plants to produce plants or plant parts, including, but not limited to leaves, stems, roots, reproductive, and seed, with a modified content of tocopherols in plant parts having transformed plant cells.
• antibodies to the protein can be prepared by injecting rabbits or mice with the purified protein or portion thereof, such methods of preparing antibodies being well known to those in the art. Either monoclonal or polyclonal antibodies can be produced, although typically polyclonal antibodies are more useful for gene isolation.
• Western analysis may be conducted to determine that a related protein is present in a crude extract of the desired plant species, as determined by cross-reaction with the antibodies to the encoded proteins. When cross-reactivity is observed, genes encoding the related proteins are isolated by screening expression libraries representing the desired plant species.
• Expression libraries can be constructed in a variety of commercially available vectors, including lambda gtl 1, as described in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
• in vitro assays are performed in insect cell cultures using baculovirus expression systems.
• baculovirus expression systems are known in the art and are described by Lee,et al. U.S. Patent Number 5,348,886, the entirety of which is herein incorporated by reference.
• expression constructs may be prepared to assay for protein activity utilizing different expression systems. Such expression constructs are transformed into yeast or prokaryotic host and assayed for dxr activity. Such expression systems are known in the art and are readily available through commercial sources.
• DNA coding sequences useful in the present invention can be derived from algae, fungi, bacteria, mammalian sources, plants, etc. Homology searches in existing databases using signature sequences corresponding to conserved nucleotide and amino acid sequences of dxr can be employed to isolate equivalent, related genes from other sources such as plants and microorganisms. Searches in EST databases can also be employed. Furthermore, the use of DNA sequences encoding enzymes functionally enzymatically equivalent to those disclosed herein, wherein such DNA sequences are degenerate equivalents of the nucleic acid sequences disclosed herein in accordance with the degeneracy of the genetic code, is also encompassed by the present invention.
• Demonstration of the functionality of coding sequences identified by any of these methods can be ca ⁇ ied out by complementation of mutants of appropriate organisms, such as Synechocystis, Shewanella, yeast, Pseudomonas, Rhodobacteria, etc., that lack specific biochemical reactions, or that have been mutated.
• the sequences of the DNA coding regions can be optimized by generesynthesis, based on codon usage, for maximum expression in particular hosts.
• the method of transformation in obtaining such transgenic plants is not critical to the instant invention, and various methods of plant transformation are cu ⁇ ently available. Furthermore, as newer methods become available to transform crops, they may also be directly applied hereunder. For example, many plant species naturally susceptible to Agrobacterium infection may be successfully transformed via tripartite or binary vector methods of Agrobacterium mediated transformation. In many instances, it will be desirable to have the construct bordered on one or both sides by T- DNA, particularly having the left and right borders, more particularly the right border. This is particularly useful when the construct uses A. tumefaciens or A. rhizogenes as a mode for transformation, although the T-DNA borders may find use with other modes of transformation. In addition, techniques of microinjection, DNA particle bombardment, and electroporation have been developed which allow for the transformation of various monocot and dicot plant species.
• included with the DNA construct will be a structural gene having the necessary regulatory regions for expression in a host and providing for selection of transformant cells.
• the gene may provide for resistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin, etc., complementation providing prototrophy to an auxotrophic host, viral immunity or the like.
• a cytotoxic agent e.g. antibiotic, heavy metal, toxin, etc.
• complementation providing prototrophy to an auxotrophic host, viral immunity or the like.
• one or more markers may be employed, where different conditions for selection are used for the different hosts.
• a vector may be used which may be introduced into the Agrobacterium host for homologous recombination with T-DNA or the Ti- or Ri-plasmid present in the Agrobacterium host.
• the Ti- or Ri-plasmid containing the T-DNA for recombination may be armed (capable of causing gall formation) or disarmed (incapable of causing gall formation), the latter being permissible, so long as the vir genes are present in the transformed Agrobacterium host.
• the armed plasmid can give a mixture of normal plant cells and gall.
• the expression or transcription construct bordered by the T-DNA border region(s) will be inserted into a broad host range vector capable of replication in E. coli and Agrobacterium, there being broad host range vectors described in the literature. Commonly used is pRK2 or derivatives thereof. See, for example, Ditta, et al, (Proc. Nat. Acad. Sci., U.S.A. (1980) 77:7347-7351) and EPA 0 120 515, which are incorporated herein by reference.
• markers which allow for selection of transformed Agrobacterium and transformed plant cells.
• a number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, kanamycin, the aminoglycoside G418, hygromycin, or the like.
• the particular marker employed is not essential to this invention, one or another marker being prefe ⁇ ed depending on the particular host and the manner of construction.
• explants For transformation of plant cells using Agrobacterium, explants may be combined and incubated with the transformed Agrobacterium for sufficient time for transformation, the bacteria killed, and the plant cells cultured in an appropriate selective medium. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be grown to seed and the seed used to establish repetitive generations and for isolation of vegetable oils.
• any means for producing a plant comprising a construct having a DNA sequence encoding the expression construct of the present invention, and at least one other construct having another DNA sequence encoding an enzyme are encompassed by the present invention.
• the expression construct can be used to transform a plant at the same time as the second construct either by inclusion of both expression constructs in a single transformation vector or by using separate vectors, each of which express desired genes.
• the second construct can be introduced into a plant which has already been transformed with the dxr expression construct, or alternatively, transformed plants, one expressing the dxr construct and one expressing the second construct, can be crossed to bring the constructs together in the same plant.
• nucleic acid sequences of the present invention can be used in constructs to provide for the expression of the sequence in a variety of host cells, both prokaryotic eukaryotic.
• Host cells of the present invention preferably include monocotyledenous and dicotyledenous plant cells.
• Baculovirus expression vectors are recombinant insect viruses in which the coding sequence for a chosen foreign gene has been inserted behind a baculovirus promoter in place of the viral gene, e.g., polyhedrin (Smith and Summers, U.S. Pat. No., 4,745,051, the entirety of which is incorporated herein by reference).
• Baculovirus expression vectors are known in the art, and are described for example in Doerfler. Curr. Top. Microbiol. Immunol. 737:51-68 (1968); Luckow and Summers, Bio/Technology 6:41-55 (1988a); Miller, Annual Review of Microbiol.
• the fungal host cell may, for example, be a yeast cell or a filamentous fungal cell.
• Methods for the expression of DNA sequences of interest in yeast cells are generally described in "Guide to yeast genetics and molecular biology", Guthrie and Fink, eds. Methods in enzymology , Academic Press, Inc. Vol 194 (1991) and Gene expression technology", Goeddel ed, Methods in Enzymology, Academic Press, Inc., Vol 185 (1991).
• Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC, Manassas, VA), such as HeLa cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells and a number of other cell lines.
• Suitable promoters for mammalian cells are also known in the art and include, but are not limited to, viral promoters such as that from Simian Virus 40 (SV40) (Fiers et al, Nature 273: 113 (1978), the entirety of which is herein incorporated by reference), Rous sarcoma virus (RSV), adenovirus (ADV) and bovine papilloma virus (BPV).
• Mammalian cells may also require terminator sequences and poly-A addition sequences. Enhancer sequences which increase expression may also be included and sequences which promote amplification of the gene may also be desirable (for example methotrexate resistance genes).
• Vectors suitable for replication in mammalian cells are well known in the art, and may include viral replicons, or sequences which insure integration of the appropriate sequences encoding epitopes into the host genome. Plasmid vectors that greatly facilitate the construction of recombinant viruses have been described (see, for example, Mackett et al, J Virol. 49: - 51 (1984); Chakrabarti et al, Mol. Cell. Biol. 5:3403 (1985); Moss, In: Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds., Cold Spring Harbor Laboratory, NN., p. 10, (1987); all of which are herein incorporated by reference in their entirety).
• 2-C-Methyl-D-erythritol with a ca 80% e.e. was synthesized according to a Duvold, et al. (1997) Tetrahedron Lett 38:4769-4772 and Duvold, et al. (1997) Tetrahedron Lett 38:6181-6184) adapted to the production of larger amounts.
• a solution of 3-methyl-2(577)-furanone (200 mg, 2 mmol) in dry ether (20 ml) was added at 0°C over a period of 15 min to a sti ⁇ ed suspension of LiAUL (46 mg, 1.2 mmol) in dry ether (20 ml) under argon.
• reaction mixture was sti ⁇ ed at O°C for further 2 h.
• a saturated solution of NtLCI (2 ml) was slowly added until the excess of LiAIIJt was destroyed.
• IM HCl solution was acidified with a IM HCl solution until all aluminum salts were dissolved, the aqueous phase was extracted with ethyl acetate (6 x 20 ml). The combined organic layers were washed with saturated brine and dried over anhydrous Na 2 SO .
• Enantioselective dihydroxylation of diacetate 300 mg, 1.6 mmol was performed by sti ⁇ ing at O°C in ter- butanol/water (1: 1. v/v, 6 ml) in the presence of the chiral osmylation reagent AD- mix-b (2.5 g) and CH 3 SO 2 NH 2 (152 mg, 1.6 mmol). After 24 hours, the reaction was quenched with solid Na2SO 3 and additional sti ⁇ ing for 30 minutes.
• EXAMPLE 2 Site-Directed Marker Insertion Mutagenesis of the -for gene of E. coli
• the region extending from the 5 '-region of the-i r gene to the 3 '-flanking region of the yaeS gene was amplified by PCR using genomic DNA isolated from the wild type E. coli strain W3110 (Kohara et al., 1987) and the primers Pl(5'- CTCTGGATGT CATATGAAGCAACTC-3' (SEQ ID NO:3); the underlined ATG co ⁇ esponds to the translation start codon of the dxr gene) and P2 (5'- CCGCATAACACCGCCAACC-3' (SEQ ID NO:4); located at the 3'-flanking region of the yaeS gene).
• the reaction mixture for the PCR was prepared in a final volume of 50 ⁇ l, containing the DNA template (100 ng), 0.5 ⁇ M of each primer, 200 ⁇ M of each deoxynucleoside triphosphate, 20 mM of Tris-HCl adjusted to pH 8.8, 2 mM Of MgSO 4 , 10 mM of KCI, 10 mM of (NH 4 ) 2 SO 4 , 0.1 mg/ml of BSA and 0.1% Triton X- 100.
• the sample was covered with mineral oil, incubated at 94°C for 3 min and cooled to 8O°C.
• Pfu DNA polymerase (1.25 units, Stratagene) was added and the reaction mixture was incubated for 30 cycles consisting of 45 sec at 94°C, 45 sec at 59°C and 10 min at 72°C, followed by a final step of 10 min at 72°C. After amplification, adenines were added to the 3' ends of the PCR product as indicated by the manufacturers protocol and the adenylated product was cloned into the pGEM-T vector (Promega), to create plasmid pMJl.
• the CAT (chloramphenicol acetyl transferase) gene present in plasmid pCAT19 was excised by digestion with Rstl and Xbal, treated with T4 DNA polymerase and cloned into the unique Asull site present in the dxr gene by blunt end ligation (after treatment with T4 DNA polymerase), resulting plasmid pMJ2. Restriction enzyme mapping was used to identify the clones in which the CAT gene was in the same orientation than the dxr gene.
• Plasmid pMJ3 was constructed by subcloning the Spel-Sphl fragment excised from plasmid pMJ2 into the Nhel-Sphl sites of plasmid pBR322. Plasmid pMJ3 was linearized by digestion with Pstl, incubated with calf intestinal alkaline phosphatase (GibcoBRL) and purified by agarose gel electrophoresis. Two ⁇ g of the purified linear plasmid pMJ3 DNA were used to transform E coli strain JC7623 (Winans et al., 1985). Transformed cells were plated onto LB plates (Ausubel et al.
• the EST database of the NCBI (dbEST) was searched with the BLASTN program using as a query the nucleotide sequence of clone MOB2 extending from nucleotides 29247 to 31317.
• Two A. thaliana EST clones (12OE8T7 and 65F11XP3', accession numbers T43949 and AA586087,respectively) containing nucleotide sequences identical to different regions of the query were found. Sequencing of the cDNA inserts revealed the two clones were overlapping.
• the longest cDNA contained an open reading frame encoding a polypeptide of 329 residues showing an identity of 41.6% (similar of 53.2%) with the C-terminal region of the E. coli DXR, thus indicating that the two cDNAs encoded truncated versions of the putative A. thaliana enzyme.
• RNA from 12-days-old light-grown Arabidopsis thaliana (var. Columbia) seedlings was purified as described (Dean et al., 1985). Rapid amplification of cDNA ends (RACE) was carried out with the 5'-RACE-System (Version 2.0) from Life Technologies/Gibco BRL, following the instructions of the supplier.
• the first strand of cDNA was synthesized using 1 ⁇ g of the RNA sample as template and the oligonucleotide DXR-GSPl (5'-ATTCGAACCAGCAGCTAGAG-3' (SEQ ID NO:6), complementary to nucleotides +767 to +786 of the sequence shown in SEQ ID NO: 1 as specific downstream primer.
• the specific downstream primer was the oligonucleotide DXR-GSP2 (5'-CCAGTAGATCCAACGATAGAG-3' (SEQ ID NO:7), complementary to nucleotides +530 to +550 of the sequence shown in SEQ ID NO: 1) and the upstream primer was the oligonucleotide 5'-RACE-AAP (supplied in the kit).
• the specific downstream primer was the oligonucleotide DXR-GSP3 (5'- GGCCATGCTGGAGGAGGTTG-3' (SEQ ID NO:8), complementary to nucleotides +456 to +475 of the sequence shown in SEQ ID NOJ) and the upstream primer was the oligonucleotide AUAP (supplied in the kit).
• DXR-GSP3 5'- GGCCATGCTGGAGGAGGTTG-3' (SEQ ID NO:8), complementary to nucleotides +456 to +475 of the sequence shown in SEQ ID NOJ
• the upstream primer was the oligonucleotide AUAP (supplied in the kit).
• the amplification process was initiated by denaturation of the sample (3 min at 94°C), cooling to 80°C and addition of Taq DNA polymerase.
• the reaction mixture of the first PCR was incubated for 15 cycles consisting of 30 sec at 94°C, 30 sec at 55°C and 1 min at 72°C, followed by a final step of 5 min at 72°C.
• the sample obtained was diluted one to ten in the reaction mixture of the second PCR and incubated for 30 cycles consisting of 30 sec at 94°C, 30 sec at 61°C and 1 min at 72°C, with a final step of 5 min at 72°C.
• the final amplification products were purified by agarose gel electrophoresis, cloned into plasmid pBluescript SK+ and sequenced (SEQ ID NO: 1).
• EXAMPLE 4 Cloning of a 1 -deoxy-D-xylulose 5-phosphate reductoisomerase cDNA from Arabidopsis thaliana
• the corresponding transcription start site was mapped by using the RACE technique.
• Primers were designed on the basis of the alignment between the DXR from E. coli and the amino acid sequence deduced from the A. thaliana genomic clone.
• the deduced amino acid sequence from the Arabidopsis dxr nucleic acid sequence (SEQ ID NOJ) is provided in SEQ ID NO:2.
• the first strand of cDNA was synthesized using RNA from A. thaliana seedlings as a template and the oligonucleotide DXR-GSPl as primer.
• This oligonucleotide was complementary to the region between positions +767 and +786 of the genomic sequence shown in SEQ ID NOJ.
• two nested PCR reactions were ca ⁇ ied out to ampl4 the 5' end of the mRNA.
• the downstream specific primers used for the first and second nested PCR reactions were complementary to the regions extending from positions +530 to +550 (primer DXR-GSP2) and +456 to +475 (primer DXR-GSP3), respectively.
• Four clones co ⁇ esponding to the major amplification product were sequenced and found to have the same 5 '-end, which co ⁇ esponds to the adenine at position +1 in the genomic sequence shown in SEQ ID NOJ.
• a cDNA containing the whole coding sequence of the Arabidopsis DXR was amplified by two consecutive PCR reactions from a cDNA library derived from the A. thaliana (var. Columbia) cell suspension line T87. An aliquot of the library was ethanol- precipitated and resuspended in water.
• the reaction mixture for the first PCR was prepared in a final volume of 25 ⁇ l containing the DNA template (equivalent to 4xl0 5 pfu of cDNA library), 0.5 ⁇ M of the upstream primer DXR-34 (5'- CAAGAGTAGTAGTGCGGTTCTCTGG-3' (SEQ ID NO:9), co ⁇ esponding to nucleotides +34 to +58 of the sequence shown in SEQ ID NOJ), 0.5 ; ⁇ M of the downstream primer DXR-E2 (5'-CAGTTTGGCTTGTTCGGATCACAG-3' (SEQ ID NOJ0), complementary to nucleotides +3146 to + 3169 of the sequence shown in SEQ ID NOJ), 200 ⁇ M of each deoxynucleoside triphosphate, 20 mM of Tris-HCI adjusted to pH 8.8, 2 mM Of MgSO 4 , 10 mM of KCI, 10 MM of (NH 4 ) 2 SO 4 , 0J mg/ml of B
• the sample was covered with mineral oil, incubated at 94°C for 3 min and cooled to 80°C.
• Pfu DNA polymerase (1.25 units, Stratagene) was added and the reaction mixture was incubated for 35 cycles consisting of 30 sec at 94°C, 40 sec at 55°C and 6.5 min at 72°C, followed by a final step of 15 min at 72°C.
• the reaction mixture was diluted one to ten with water and 5 ⁇ l were used as a template for the second PCR that was performed using the same conditions as described for the previous amplification, except that the volume of the reaction mixture was increased to 50 ⁇ l and the number of cycles was reduced to 15.
• the amplification product was purified by agarose gel electrophoresis and cloned into plasmid pBluescript SK+. The resulting plasmid was named pDXR-At.
• a cDNA clone encoding the entire A. thaliana DXR was obtained by PCR from a cDNA library using primers DXR-34 and DXR-E2 co ⁇ esponding to the regions extending from positions +34 to +58 and +3146 to +3169 of the genomic sequence, respectively.
• the identity of the amplified cDNA was confirmed by DNA sequencing, The alignment of the cDNA and the genomic sequences showed that the A. thaliana DXR gene contains 12 exons and 11 introns which extend over a region of 3.2 Kb (SEQ ID NOJ).
• the cloned cDNA encodes a protein of 477 amino acid residues with a predicted molecular mass of 52 kDa.
• the alignment of A. thaliana and E. coli DXR ( Figure 1) reveals that the plant enzyme has a N-terminal extension of 79 residues with the typical features of plastid transit peptides (von Heijne et al., 1989). The two proteins show an identity of 42.7% (similarity of 54.3%).
• the region of the DXR cDNA encoding amino acid residues 81 to 477 was amplified by PCR from plasmid pDXR- At and cloned into a modified version of plasmid pBAD-GFPuv (Clontech). In this plasmid, expression is driven by the P BAD promoter which can be induced with arabinose and repressed with glucose.
• plasmid pBAD-GFPuv was modified by removing the Ndel site located between pBR322ori and the araC coding region (position 4926-4931) by site-directed mutagenesis following the method of Kunkel et al.
• oligonucleotide pBAD-mutl (5'- CTGAGAGTGCACCATCTGCGGTGTGAAATACC-3' (SEQ ID NO: 11)) was used as mutagenic primer.
• the resulting plasmid was designated pBAD-Mi.
• N ⁇ 7el and EcoRl restriction sites were introduced at appropriate positions of the A. thaliana DXR cD ⁇ A by PCR, using the plasmid pDXR-At as template and the oligonucleotides 5'-MVKPI (5'-
• GGCATATGGTGAAACCCATCTCTATCGTTGGATC-3' (S ⁇ Q ID ⁇ O.J2), complementary to nucleotides +522 to +544 of the sequence shown in S ⁇ Q ID NOJ; the underlined sequence contains the Ndel site) and DXR- ⁇ ND(5'- ACGAATTCATTATGCATGAACTGGCCTAGCACC-3' (SEQ ID NO: 13), complementary to nucleotides+2997 to +3018 of the sequence shown in SEQ ID NO: 1 ; the underlined sequence contains the EcoRl site) as mutagenic primers.
• the PCR amplification product was digested with N-M and EcoRl and cloned into plasmid pBAD-Ml digested with the same restriction enzyme. This resulted in the substitution of the GFPuv coding sequence in plasmid pB AD-MI by the co ⁇ esponding coding sequence of the.
• A. thaliana DXR The resulting plasmid, designated pBAD-DXR, was introduced into strain XLl-Blue. Plasmid pBAD-Ml, encoding GFPuv, was used as a control in the complementation studies.
• the function of the cloned A. thaliana DXR has been established by complementation analysis of an E. coli strain carrying a disruption in the dxr gene (strain JC7623dxr.:CAT) (see Example 2). This strain requires 2-C-methyl-D- erythritol (ME) for growth.
• ME 2-C-methyl-D- erythritol
• the appropriate cDNA fragment was cloned into a derivative of plasmid pBAD-GFPuv, under the control of the PBAD promoter, and the resulting plasmid (pBAD-DXR) introduced into the JC7623dxr.- CAT strain.
• Expression from the PBAD promoter is inducible by arabinose and repressed by glucose. Induction with arabinose allows growth of strain JC7623dxr.
• - CAT harbouring plasmid PBAD-DXR in the absence of ME, whereas no growth was observed in the presence of glucose.
• Strain JC7623dxr.,:CAT carrying the control plasmid pB AD-MI does not grow in the presence of arabinose on medium lacking ME.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• General Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Polymers & Plastics (AREA)
• Medicinal Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Physics & Mathematics (AREA)
• Nutrition Science (AREA)
• Plant Pathology (AREA)
• Biophysics (AREA)
• Food Science & Technology (AREA)
• Cell Biology (AREA)
• General Chemical & Material Sciences (AREA)
• Animal Husbandry (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)
• Enzymes And Modification Thereof (AREA)
• Saccharide Compounds (AREA)
• Pyrane Compounds (AREA)

Applications Claiming Priority (5)

Publications (2)

Family

ID=26828010

Family Applications (2)

Family Applications Before (1)

Country Status (14)

Cited By (12)

Families Citing this family (65)

Family Cites Families (85)

• 2000
  • 2000-04-14 AU AU42493/00A patent/AU776316B2/en not_active Expired
  • 2000-04-14 WO PCT/US2000/010367 patent/WO2000063389A1/fr active IP Right Grant
  • 2000-04-14 DE DE60025713T patent/DE60025713T2/de not_active Expired - Lifetime
  • 2000-04-14 DK DK00922287T patent/DK1190068T3/da active
  • 2000-04-14 US US09/549,848 patent/US6541259B1/en not_active Expired - Lifetime
  • 2000-04-14 CA CA2370616A patent/CA2370616C/fr not_active Expired - Lifetime
  • 2000-04-14 MX MXPA01010488A patent/MXPA01010488A/es active IP Right Grant
  • 2000-04-14 AT AT00923461T patent/ATE358723T1/de active
  • 2000-04-14 PT PT00922287T patent/PT1190068E/pt unknown
  • 2000-04-14 JP JP2000612468A patent/JP2002541851A/ja active Pending
  • 2000-04-14 BR BR0010616-0A patent/BR0010616A/pt not_active Application Discontinuation
  • 2000-04-14 DE DE60034224T patent/DE60034224T2/de not_active Expired - Lifetime
  • 2000-04-14 JP JP2000612470A patent/JP2003527077A/ja active Pending
  • 2000-04-14 AU AU43579/00A patent/AU777584B2/en not_active Expired
  • 2000-04-14 ES ES00922287T patent/ES2253220T3/es not_active Expired - Lifetime
  • 2000-04-14 EP EP00922287A patent/EP1190068B1/fr not_active Expired - Lifetime
  • 2000-04-14 CA CA2369844A patent/CA2369844C/fr not_active Expired - Lifetime
  • 2000-04-14 CN CNB008087849A patent/CN1318593C/zh not_active Expired - Lifetime
  • 2000-04-14 WO PCT/US2000/010368 patent/WO2000063391A2/fr active IP Right Grant
  • 2000-04-14 CN CNB008084572A patent/CN100387721C/zh not_active Expired - Lifetime
  • 2000-04-14 EP EP00923461A patent/EP1171610B1/fr not_active Expired - Lifetime
  • 2000-04-14 BR BRPI0009763A patent/BRPI0009763B8/pt active IP Right Grant
  • 2000-04-14 AT AT00922287T patent/ATE316578T1/de not_active IP Right Cessation
  • 2000-04-14 MX MXPA01010486A patent/MXPA01010486A/es active IP Right Grant
• 2001
  • 2001-11-13 US US09/987,025 patent/US7067647B2/en not_active Expired - Lifetime
• 2003
  • 2003-01-23 US US10/349,508 patent/US7265207B2/en not_active Expired - Lifetime
  • 2003-05-13 US US10/437,169 patent/US7141718B2/en not_active Expired - Lifetime
• 2004
  • 2004-10-15 US US10/966,829 patent/US7335815B2/en not_active Expired - Lifetime
• 2005
  • 2005-01-21 AU AU2005200270A patent/AU2005200270B2/en not_active Expired

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events